Upregulation of the hypothalamo-neurohypophysial system and activation of vasopressin neurones attenuates hyperalgesia in a neuropathic pain model rat

Arginine vasopressin (AVP) is a hypothalamic neurosecretory hormone well known as an antidiuretic, and recently reported to be involved in pain modulation. The expression kinetics of AVP and its potential involvement in the descending pain modulation system (DPMS) in neuropathic pain (NP) remains unclear. We investigated AVP expression and its effects on mechanical and thermal nociceptive thresholds using a unilateral spinal nerve ligation (SNL) model. All rats with SNL developed NP. Intensities of enhanced green fluorescent protein (eGFP) in the supraoptic and paraventricular nuclei, median eminence, and posterior pituitary were significantly increased at 7 and 14 days post-SNL in AVP-eGFP rats. In situ hybridisation histochemistry revealed significantly increased AVP mRNA expression at 14 days post-SNL compared with the sham control group. The chemogenetic activation of AVP neurones significantly attenuated mechanical and thermal hyperalgesia with elevated plasma AVP concentration. These analgesic effects were suppressed by pre-administration with V1a receptor antagonist. AVP neurones increased the neuronal activity of serotonergic dorsal raphe, noradrenergic locus coeruleus, and inhibitory interneurones in the spinal dorsal horn. These results suggest that the hypothalamo-neurohypophysial system of AVP is upregulated in NP and activated endogenous AVP exerts analgesic effects via the V1a receptors. AVP neurones may activate the DPMS.

AVP neurones activate serotonergic neurones in the DR, noradrenergic neurones in the LC and paired box gene-2 positive inhibitory interneurones in the spinal dorsal horn. The withdrawal threshold of the CNO group was significantly elevated, from 30 to 120 min, compared with that of the Saline group in untreated AVP-hM3Dq-mCherry Tg rats (p = 0.009, 0.001, 0.006, and 0.005 at 30, 60, 90, and 120 min, respectively) (Fig. 7Aa). The withdrawal latency of the CNO injection group was significantly prolonged from 60 to 120 min, compared with that of the Saline group (p = < 0.001, 0.048, and 0.001 at 60, 90, and 120 min, respectively) (Fig. 7Ab). The number of tryptophan hydroxylase (TPH)-and Fos-ir double positive neurones were significantly increased in the CNO group in the ventrolateral part of DR (DRvl), the dorsal part of DR (DRd), ventral part of DR (DRv), compared with the Saline group (DRvl, p < 0.001; DRd, p < 0.001; DRv, p < 0.001) (Fig. 7Ba,b). The tyrosine hydroxylase (TH)-and Fos-ir double positive neurones were significantly increased in the CNO group in the LC, compared with the Saline group (p < 0.001) (Fig. 7Ca,b). The number of paired box gene-2 (PAX-2) and Fos-ir colocalized neurones were significantly increased in the CNO group in the laminae I-II and III-IV of L5 ipsilateral dorsal horn, compared with the Saline group (laminae I-II, p < 0.001; laminae III-IV, p < 0.001) (Fig. 7Da,b).

Discussion
The results of this study are summarised as follows: (1) the fluorescence intensity of eGFP in the SNL group was significantly increased in the SON, PVN, ME, and PP; (2) the expression level of AVP mRNA in the SON and PVN and POMC mRNA in the AP was significantly increased, whereas that of CRH mRNA in the pPVN was significantly decreased; (3) the activation of endogenous AVP neurones by CNO administration led to an analgesic effect; (4) the analgesic effect of endogenous AVP was significantly suppressed by pre-treatment with a V1a receptor antagonist; (5) serotonergic neurones in the DR, noradrenergic neurones in the LC and PAX-2 positive interneurones in the spinal dorsal horn were found to be activated after stimulation of endogenous AVP neurones. AVP produced in the mPVN and SON is transported to the PP via the inner layer of the median ridge and secreted into the systemic circulation. Despite increased AVP-eGFP and AVP mRNA expression in the magnocellular region, the plasma AVP concentration was not significantly increased (see Supplementary Fig. S1 online), and mechanical and thermal hyperalgesia were still observed in the SNL. Although the expression of www.nature.com/scientificreports/ www.nature.com/scientificreports/ AVP-eGFP, AVP mRNA, and plasma AVP concentration was significantly increased, hyperalgesia persisted in our previous AA model and knee OA model 20,22 . We previously reported that AVP and Oxytocin (OXT) exhibited similar kinetics in various pain models 20,21,25 . OXT-mRFP1 fluorescence intensity in the SON, mPVN and PP was significantly increased in the NP model rats using OXT-mRFP1 Tg rats in which OXT was labelled with red fluorescent protein, but plasma OXT concentration remained unchanged 26 . The half-life of blood AVP and OXT is very short and their plasma concentrations may not correlate with their activity in the brain 27 . Therefore, it seems possible that NP activates the synthesis of AVP without stimulating its secretion. The SNL group exhibited a significant increase in the fluorescence intensity of AVP-eGFP and AVP mRNA expression in the pPVN, and CRH mRNA levels in the pPVN were significantly decreased at day 14 after SNL. AVP and CRH secreted from the parvocellular region of the PVN are secreted from axon terminals in the outer layer of the median ridge to the pituitary portal vein. Secretion of adrenocorticotropic hormone (ACTH) from the AP activates the hypothalamo-pituitary-adrenal (HPA) axis 28 . CRH and AVP have a secretagogue effect on the ACTH and work synergistically in the HPA axis 29 . In a previous study, chronic pain induced by AA displayed dysfunction of the HPA axis 30,31 . Indeed, AVP, but not CRH, plays a key role in promoting ACTH secretion under chronic stress conditions 30 . In our previous study, AVP mRNA levels in the pPVN were significantly increased, and CRH mRNA was significantly decreased in a chronic pain model of AA 20 . In the present study, we showed that AVP might be a key modulator in the HPA axis of the NP model. SR49059 is a V1a receptor antagonist, but may also act on V1b and OXT receptors. It was reported that SR49059 had an affinity more than two orders of magnitude lower for V1b and OXT receptors and did not exhibit intrinsic agonist activity 32 . On the other hand, it has been reported that 3-5% of neurones in the magnocellular of the hypothalamus co-express AVP and OXT 33 . Our previous report also showed OXT immunostaining positivity in SON (3.57%) and PVN (2.17%) mCherry neurones in AVP-hM3Dq-mCherry Tg rats 24 . Hence, i.p. CNO administration may activate some OXT neurones in the hypothalamus, which may be involved in pain modulation. However, although the plasma OXT concentration was not measured in the present study, the proportion of neurones co-expressing OXT and AVP was only a few percent, which suggests that the effect may be small. Accordingly, our data suggest that endogenous AVP is involved in pain modulation by central and peripheral V1a receptors. In the central nervous system, microinjection of AVP into the amygdala, caudate nucleus, and nucleus accumbens increases pain thresholds [34][35][36] . It has also been suggested that the analgesic mechanism of AVP in the central nervous system may be involved in endogenous opioid and serotonin systems 37 . In addition, a projection from the hypothalamus to the spinal dorsal horn involved in analgesia via V1a receptors is thought to exist 38 . In the peripheral nervous system, a previous study showed that AVP upregulates the function of GABA A in dorsal root ganglion neurones 39 . Furthermore, DNA microarray analysis revealed that the V1a receptor was upregulated in a mouse model of NP 40 . Subcutaneous plantar injection of AVP was shown to attenuate formalininduced nociceptive pain via peripheral V1a receptors 6 .
Furthermore, the activation of endogenous AVP neurones increased the neuronal activity of the serotonergic DR neurones, noradrenergic LC neurones and inhibitory interneurones of the spinal dorsal horn involved in the descending pain inhibitory system. The DR and LC are important neuronal nuclei involved in the DPMS and these nuclei project to the spinal dorsal horn 15,[41][42][43][44] . The activation of the DR and LC releases serotonin and noradrenaline, respectively 41 . AVP neurones project to the DR and activate serotonergic neurones via V1a receptors 45,46 . Moreover, AVP neurones also project to the LC, and V1a and V1b receptors are located on noradrenergic neurones, resulting in noradrenergic neuronal activation 14,45,47 . Thus, chemogenetic activation of AVP neurones in AVP-hM3Dq-mCherry-Tg rats may activate serotonergic and noradrenergic neurones, which in turn activate PAX-2 positive inhibitory interneurones in the spinal dorsal horn.
This study has several limitations. AVP neurones have been located in the central nervous system in the piriform cortex, olfactory bulb, retina, and LC using AVP-eGFP Tg rats 48 . Administration of CNO could also activate AVP neurones in these regions, but we have not confirmed in the present study whether hM3Dq is expressed in regions other than the hypothalamus. However, the main source of AVP production is likely to be the hypothalamic SON, PVN, and suprachiasmatic nucleus (SCN) 48 . AVP production by non-hypothalamic AVP neurones is considered to be small. Also, we did not perform CNO administration experiments using Wistar rats that do not express hM3Dq. Clozapine is the parent compound of CNO, which has been demonstrated to be converted to clozapine 49 . I.p. administration of clozapine (30 mg/kg) was reported to activate hypothalamic vasopressin neurones 50 . In our previous studies, it was confirmed that i.p. administration of CNO (1 mg/kg) to Wistar rats did not significantly increase the number of c-Fos positive neurones in the SON and PVN, nor plasma AVP concentrations 24 . Therefore, the pharmacological action of CNO itself on the AVP system is considered to be poor in rats that do not express hM3Dq. The c-Fos positive neurones in the DR and LC may contain fibres of the ascending pain modulation system; however, it is difficult to distinguish between neurones of the ascending pain modulation system and those of the DPMS. Nevertheless, our study showed that the number of inhibitory interneurone-specific PAX-2 positive neurones in the spinal dorsal horn, which is the end point of the descending pathway, was increased, which may indicate that chemogenetic activation of endogenous AVP neurones stimulated the DPMS. Besides, PAX-2-positive neurones may be activated by pathways other than the descending pain-inhibitory system, such as the dorsal root ganglia. Further studies using AVP-hM3Dq-mCherry Tg rats are required.
In conclusion, the results of this study suggest that the hypothalamo-neurohypophysial system of AVP is upregulated in a rat model of NP and that activated endogenous AVP exerts an analgesic effect via the V1a receptor and may activate the DPMS.

Methods
Animals. AVP-eGFP Tg rats (7 weeks old, weighing 238-305 g) and AVP-hM3Dq-mCherry Tg rats (7 weeks old, weighing 253-350 g) were bred and maintained as described previously 22,24,51 . All procedures were performed according to the guidelines on the use and care of laboratory animals established by the Physiological Society of Japan. All experiments performed in this study were approved by the Ethics Committee on Animal Care and Experimentation (permission number: AE10-012) of the University of Occupational and Environmental Health, Japan. This research was conducted in accordance with ARRIVE guidelines.
Spinal nerve ligation procedure. AVP-eGFP Tg and AVP-hM3Dq-mCherry Tg rats were randomly divided into three groups: Control, Sham and SNL. Rats in the Control group were untreated, whereas rats in the sham surgery (Sham) group were anaesthetised through subcutaneous injection with a combination of anaesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol), and the left lumbar 5 (L5) spinal nerve was disclosed without ligation. Rats in the SNL model group were anaesthetised, and the left L5 spinal nerve was ligated using 6-0 silk sutures 54 .

Measurement of mechanical and thermal sensitivities. The mechanical and thermal nociceptive
thresholds were evaluated at the baseline (BL, before the SNL procedure) and at postoperative days 7 and 14 in AVP-eGFP Tg rats. Before evaluating the mechanical and thermal nociceptive thresholds, rats were acclimatised to the experimental environment for at least 30 min. Thereafter, we performed the manual von Frey test on the left hind paw using calibrated von Frey filaments (North Coast Medical, Gilroy, CA, USA) and a hot plate test (52.5 °C) as described previously 22 . The mechanical and thermal nociceptive thresholds were determined according to previously described methods 21,22,26 . Tissue preparation. At  Expression level of AVP-eGFP in the SON, PVN, ME, and PP. Digital images of the SON, pPVN, mPVN, ME, PP, and spinal cord were captured using fluorescence microscopy with a GFP filter (ECLIPS E 600; Nikon Corporation) to measure the fluorescence intensities of AVP-eGFP 22 . Two sections, each including SON, PVN, and ME, were selected for image analysis. We calculated the mean fluorescence intensity value for the eGFP per unit area in the SON, pPVN, mPVN, iME, oME, and PP for each animal group on postoperative days 7 and 14 using NIS Elements software (Nikon Corporation).
In situ hybridisation histochemistry for AVP, CRH, and POMC mRNA. AVP-eGFP Tg rats (n = 7 in each group) were decapitated at postoperative day 14 for in situ hybridisation histochemistry. The brains obtained after decapitation were placed in a deep freezer at − 80 °C. Coronal sections of the brains were cut into a thickness of 12 μm using a cryostat (OTF5000, Bright Instrument Co, Ltd., England) at − 20 °C and mounted onto glass slides coated with gelatin/chrome alum. Two sections of the SON and PVN, and six sections of the AP were selected to measure the density of the autoradiograph. Subsequently, we used a 35 S-labelled oligodeoxynucleotide probe for AVP, CRH, and POMC mRNA analysis 56  Plasma AVP concentrations. We collected the trunk blood of AVP-hM3Dq-mCherry Tg rats decapitated after the behavioural experiment at postoperative day 15 after SNL surgery. Plasma samples were obtained by centrifugation for 10 min at 4 °C for 1000g and measured in duplicate. Plasma AVP concentrations were determined by radioimmunoassay with specific anti-AVP antibodies 57 . The results were averaged for each group.

Evaluation of the activity of serotonergic DR neurones and noradrenergic LC neurones and inhibitory interneurones of the spinal dorsal horn.
Untreated AVP-hM3Dq-mCherry Tg rats were divided into the CNO injection group and the Saline injection group. Mechanical and thermal nociceptive thresholds were measured from baseline to 180 min after CNO or saline i.p. administration. Two weeks after evaluation of pain threshold, perfusion fixation was performed at 90 min after CNO or saline injection, and the brain and spinal cord were removed; three sections of the DR and LC and L5 spinal dorsal horn were prepared as described in "Tissue preparation". For double immunohistochemistry, each three brain sections were incubated for 72 h at 4 °C with a goat primary c-Fos antibody (sc-52G; Santa Cruz Biotechnology, Dallas, TX, USA; 1:250) and a rabbit primary TPH antibody (Bioss Antibodies, Woburn, MA, USA; 1:100) or a rabbit primary TH antibody (GeneTex, Irvine, CA, USA; 1:200) in PBST with 5% normal donkey serum. Three spinal cord slices were incubated with a rabbit primary c-Fos antibody (Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000) and a mouse primary PAX-2 antibody (Abnova, Taipei, Taiwan; 1:500) in PBST with 5% normal donkey serum. After three washes with PBST for a total of 30 min, the slices were incubated for 24 h at 4 °C with a secondary antibody solution. Sections were washed three times in 0.1 M PBS for a total of 30 min. The images of DR and L5 spinal dorsal horn were observed using a BZ-X800 All-in-One fluorescence microscope (Keyence, Osaka, Japan). The sections of LC were observed using Nikon fluorescence microscope. The number of co-expressing neurones in each of the three sections was counted and averaged.
Statistics and reproducibility. The data are shown as the mean ± standard error of the mean (SEM), and the mean deviation from the control rats (%) ± SEM were calculated from the results. All data were analysed using one-way analysis of variance with Bonferroni's post hoc test for multiple comparisons. The unpaired Student's t-test was used based on the results of the Shapiro-Wilk test for comparison between the two groups (Stata/IC 16; StataCorp, College Station, TX, USA). Statistical significance was set at p < 0.05. Each experiment was carried out independently at least twice, with similar results. The sample size for this experiment was determined on the basis of previous reports which showed significant effects.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/